P999: ERK1/2 INHIBITION REDUCES OSTEOPONTIN PLASMA LEVELS AND BONE MARROW FIBROSIS IN A MYELOFIBROSIS MOUSE MODEL
Authors: S. Rontauroli, E. Bianchi, L. Tavernari, M. Dall’Ora, G. Grisendi, M. Mirabile, S. Sartini, E. Genovese, C. Carretta, S. Mallia, S. Parenti, L. Fabbiani, N. Bartalucci, L. Losi, M. Dominici, A. M. Vannucchi, R. Manfredini
Journal: HemaSphere (2022)

Background: Primary myelofibrosis (PMF) is a stem cell disorder belonging to Philadelphia-negative myeloproliferative neoplasms (MPNs). The disruption of bone marrow (BM) microenvironment due to the extensive deposition of extracellular matrix fibers is the distinctive trait of PMF and is accompanied by hematopoietic stem cells (HSCs) mobilization and extramedullary hematopoiesis. BM fibrosis is caused by the complex interaction between stromal and hematopoietic cells belonging to the neoplastic clone, in particular megakaryocytes and monocytes play a pivotal role through the production of pro-fibrotic cytokines. Supporting this hypothesis, we have previously demonstrated that osteopontin (OPN) contribute to the development of BM fibrosis. OPN plasma levels are increased in PMF patients and correlate with higher BM fibrosis grade, circulating CD34+ cells and inferior survival. Megakaryocytes and monocytes turned out as the main source for OPN production that can induce fibroblasts and mesenchymal stromal cells proliferation, as well as collagen upregulation. Aims: To determine whether OPN might represent a druggable target in PMF we assessed the inhibition of signaling pathways responsible for its production. In particular, we evaluated the effect of ERK1/2 inhibition over OPN expression and myelofibrosis development in a MF mouse model. Methods: The activity of inhibitors of signaling pathways affecting OPN expression was evaluated in vitro in human primary monocytes, given their key role in OPN secretion. The effects on cell viability were assessed by XTT assay at 72 hours of treatment, while OPN expression and production were evaluated by qRT-PCR and ELISA, respectively, at 72 and 96 hours of treatment. Inhibitors of OPN production selected from the in vitro screening were analyzed for their effect on OPN plasma levels in a MF mouse model induced by the treatment with a thrombopoietin receptor agonist Romiplostim (Rom). Hematological parameters and splenomegaly were monitored over time while BM and spleen fibrosis were evaluated at sacrifice. Results: Our in vitro analysis demonstrated that drug inhibitors of ERK1/2, MEK1/2, p38 and a Ca2+ channel antagonist were able to reduce OPN expression in primary monocytes, both at RNA and protein levels, without affecting cell viability. Next, we moved to a MF mouse model obtained through the stimulation of thrombopoietin receptor that develops bone marrow and spleen fibrosis, together with increased OPN expression, faster than JAK2V617F knock-in mice. ERK1/2 inhibition by Ulixertinib did not affect the development of thrombocytosis and splenomegaly but was able to significantly reduce OPN plasma levels. Interestingly, ERK1/2 inhibition also counteracted the development of bone marrow fibrosis in Rom-treated mice. The same results were observed in mice when Ulixertitnib was given together with the JAK inhibitor Ruxolitinib. Drug combination resulted in a reduction of both OPN plasmatic levels and bone marrow fibrosis development. Summary/Conclusion: Our results demonstrated that Ulixertinib treatment reduced OPN production both in vitro and in vivo. Moreover, in a MF mouse model, the concurrent inhibition of ERK1/2 and JAK2 signaling pathways displayed synergistic effects by diminishing OPN plasma levels and constraining BM fibrosis. These results provide a rational for the development of novel combination therapeutic approach for PMF patients.